A serious shortcoming of current gene therapy strategies and conventional methods of introducing macromolecules, particularly nucleic acids, into cells is the inability of vector and delivery system combinations to deliver nucleic acids efficiently into the interior of cells of a targeted population. Methods for introducing macromolecules, particularly nucleic acid, into a single cell or group of cells are varied. Methods commonly used include chemical treatments, liposome mediated transfection, microinjection, electroporation and particle bombardment. However, these techniques can be time-consuming and suffer from low yields or poor cell survival, and not all cell types are amenable to these methods of introducing macromolecules into a cell.
Many compositions and methods are known for delivering a nucleic acid to an animal cell or tissue. Such compositions include “naked” (i.e. non-complexed) nucleic acids, nucleic acids complexed with cationic molecules such as polylysine or liposome-forming lipids, and virus vectors. Naked nucleic acids can be taken up by various animal cells, but are subject to nucleolysis, both inside and outside of cells that take them up. Nucleic acid analogs which are relatively resistant to nucleolysis, including phosphorothioate nucleic acid analogs, are used to overcome nucleolysis. However, when incorporation of the nucleic acid into the genome of the target cell is desired, the use of nucleic acid analogs are of limited use.
Numerous vectors comprising a nucleic acid complexed with a compound to improve stability or uptake of the nucleic acid by a target cell have also been described. Such compounds include, for example, calcium phosphate, polycations such as diethylaminoethyl-dextran, polylysine, or polybrene, and liposome-forming lipids such as didocylmethylammonium bromide and Lipofectamine®. However, traditional transfections with a DNA vector complexed with another composition severely limit the ability to control the amount of mRNA transcription or protein expression, resulting in unnatural levels of protein expression which cannot otherwise be controlled.
Virus vectors are generally regarded as the most efficient nucleic acid vectors. Recombinant replication-defective virus vectors are often used to transduce (i.e., infect) animal cells. Such vectors have included retrovirus, adenovirus, adeno-associated virus vectors, and herpesvirus vectors. While highly efficient for gene transfer, a major disadvantage associated with the use of virus vectors is the inability of many virus vectors to infect non-dividing cells, limiting the cell types that can be transfected. Further, if integration of a nucleic acid into the genome is not desired, certain viruses, such as retrovirus vectors, are not recommended. Further, there is often a size limit to the length of the gene or cDNA that can be introducted into a vector. In addition, virus gene vectors do not permit appropriate regulation of gene expression over time in transfected cells.
Despite the development and refinement of the techniques discussed above, there remains a need in the art for methods and compositions which can be used to enhance the delivery of a nucleic acid to a desired cell which is to be transfected with the nucleic acid. Further, the techniques discussed above are limited to delivering one or only a few nucleic acids to study the expression of these limited numbers of nucleic acids on a cellular phenotype. The present invention provides novel method for delivering nucleic acids to a cell, and for determining the effect of multigenic nucleic acid expression on a cell.